Platelets are particles found in whole blood which participate in the process of thrombus formation and blood coagulation. A membrane bound glycoprotein, commonly known as GP llbllla, is present on the exterior surface of platelets. Glycoprotein llbllla is a non-covalent, calcium ion dependent heterodimer complex comprised of alpha and beta subunits (Jennings, et al., J. Biol. Chem. (1982) 257, 10458) and contributes to normal platelet function through interactions with Arg-Gly-Asp containing proteins such as fibrinogen. The interaction of GP llbllla with fibrinogen is stimulated by certain factors released or exposed when a blood vessel is injured. Multiple factors, including a variety of physiologic stimuli and soluble mediators, initiate platelet activation via several pathways. These pathways have a common final step which is the activation of the GP llbllla receptor on the platelet surface and its subsequent binding to fibrinogen followed by aggregation and thrombus formation. By virtue of these interactions GP llbllla is a component of the platelet aggregation system (Pytela et al., Science (1986) 231, 1559). Thus, inhibition of the interaction of GP llbllla with Arg-Gly-Asp containing ligands such as fibrinogen is a useful means of modulating thrombus formation. An inhibitor which prevents this binding interaction would antagonize platelet activation by any stimulus and therefore would have important antithrombotic properties.
Many common human disorders are characteristically associated with a hyperthrombotic state leading to intravascular thrombi and emboli. These are a major cause of medical morbidity, leading to infarction, stroke and phlebitis and of mortality from stroke and pulmonary and cardiac emboli. Patients with atherosclerosis are predisposed to arterial thromboembolic phenomena for a variety of reasons. Atherosclerotic plaques form niduses for platelet plugs and thrombii that lead to vascular narrowing and occlusion, resulting in myocardial and cerebral ischemic disease. This may happen spontaneously or following procedures such as angioplasty or endarteroectomy. Thrombii that break off and are released into the circulation cause infarction of different organs, especially the brain, extremities, heart and kidneys.
In addition to being involved in arterial thrombosis platelets may also play a role in venous thrombosis. A large percentage of such patients have no antecedent risk factors and develop venous thrombophlebitis and subsequent pulmonary emboli without a known cause. Other patients who form venous thrombi have underlying diseases known to predispose to these syndromes. Some of these patients may have genetic or aquired deficiencies of factors that normally prevent hypercoagulability, such as antithrombin-3. Others have mechanical obstructions to venous flow, such as tumor masses, that lead to low flow states and thrombosis. Patients with malignancy have a high incidence of thrombotic phenomena for unclear reasons. Antithrombotic therapy in this situation with currently available agents is dangerous and often ineffective.
Patients whose blood flows over artificial surfaces, such as prosthetic synthetic cardiac valves or through extracorporeal perfusion devices, are also at risk for the development of platelet plugs, thrombii and emboli. It is standard practice that patients with artficial cardiac valves be chronically anti-coagulated. However, in all instances, platelet activation and emboli formation may still occur despite adequate anticoagulation treatment.
Thus a large category of patients, including those with atherosclerosis, coronary artery disease, artificial heart valves, cancer, and a history of stroke, phlebitis, or pulmonary emboli, are candidates for limited or chronic antithrombotic therapy. The number of available therapeutic agents is limited and these, for the most part, act by inhibiting or reducing levels of circulating clotting factors. These agents are frequently not effective against the patient's underlying hematologic problem, which often concerns an increased propensity for platelet aggregation and adhesion. They also cause the patient to be susceptible to abnormal bleeding. Available antiplatelet agents, such as aspirin, inhibit only part of the platelet activation process and are therefore often inadequate for therapy.
An agent which effectively inhibits the final common pathway of platelet activation, namely fibrinogen binding to the GP llbllla receptor, should accordingly be useful in a large group of disorders characterized by a hyperthrombotic state as described above. The present invention contemplates such an agent which is a new composition, namely a cyclic polypeptide consisting in part of natural amino acids and in part of unnatural amino acids. This new composition interferes with the interaction of Arg-Gly-Asp containing peptides, particularly fibrinogen, with the GP llbllla complex thereby preventing platelet aggregation. Platelet aggregation has been identified as an early step in the formation of platelet plugs, emboli and thrombii in the circulatory system which in turn have been shown to play an active role in cardiovascular complications and disease. Inhibition of fibrinogen binding to the GP llbllla complex has been shown to be an effective antithrombotic treatment in animals (H. K. Gold, et al., Circulation (1988) 77, 670-677; T. Yasuda, et al., J. Clin. Invest. (1988) 81, 1284-1291; B. S. Coller, et al., Blood (1986) 68, 783-786.)
Other proteins such as fibronectin contain the Arg-Gly-Asp sequence of amino acids. Large polypeptide fragments of fibronectin have been shown to have activity for cell attachment to various surfaces which has been disclosed in U.S. Pat. Nos. 4,517,686; 4,589,881; and 4,661,111. These large polypeptides contain the amino acid sequence Arg-Gly-Asp-Ser in the interior portion of the polypeptide chain. Short peptides derived from the large polypeptides were also found to promote cell attachment to various substrates when bound on the substrate. Alternatively, the same short peptides were found to inhibit cell attachment to the same substrates when dissolved or suspended in the medium surrounding the substrate. This activity has been disclosed in U.S. Pat. Nos. 4,578,079 and 4,614,517. The short peptides were defined as EQU Q-Arg-Gly-Asp-AA1-B
wherein Q is hydrogen or an amino acid; AA1 is serine, threonine, or cysteine; and B is hydroxy or an amino acid.
A number of synthetic peptides have been disclosed as inhibitors of fibrinogen binding to platelets all of which contain the Arg-Gly-Asp sequence. See U.S. Pat. No. 4,683,291; EP 0 319 506 A2; Plow et al., Proc. Natl. Acad. Sci. USA (1985) 8 2, 8057-8061; Ruggeri et al., Proc. Natl. Acad. Sci. USA (1986) 83, 5708-5712; Haverstick et al., Blood (1985) 66, 946-952; Plow et al., Blood (1987) 70, 110-115; and references cited in the above publications.